为DGC（dystrophin-glycoprotein complex）的成分编码的基因所发生的突变会导致不同形式的肌肉萎缩。尽管对这种复合物已经做过广泛研究，但它的病理作用仍不确定。当DGC中有一个缺陷时，肌肉退化和失去运动能力也会出现在线虫 Caenorhabditis elegans中。由于有了强大的基因工具，线虫成为一种重要的动物模型。对线虫所做的基因筛选显示了一个名为snf-6的基因，它为与DGC突变体中的缺陷相同的运动缺陷编码；此外，它还为一个新的乙酰胆碱/胆碱运输子编码。这一发现为我们指出了肌肉萎缩发病机制中的一个可能的因素：不正确的清除乙酰胆碱可能会引起肌肉的过度刺激和逐渐退化。
SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans
Muscular dystrophies are among the most common human genetic diseases and are characterized by progressive muscle degeneration. Muscular dystrophies result from genetic defects in components of the dystrophin–glycoprotein complex (DGC), a multimeric complex found in the muscle cell plasma membrane. The DGC links the intracellular cytoskeleton to the extracellular matrix and is thought to be important for maintaining the mechanical integrity of muscles and organizing signalling molecules. The exact role of the DGC in the pathogenesis of disease has, however, remained uncertain. Mutations in Caenorhabditis elegans DGC genes lead to specific defects in coordinated movement and can also cause muscle degeneration. Here we show that mutations in the gene snf-6 result in phenotypes indistinguishable from those of the DGC mutants, and that snf-6 encodes a novel acetylcholine/choline transporter. SNF-6 mediates the uptake of acetylcholine at neuromuscular junctions during periods of increased synaptic activity. SNF-6 also interacts with the DGC, and mutations in DGC genes cause a loss of SNF-6 at neuromuscular junctions. Improper clearing of acetylcholine and prolonged excitation of muscles might contribute to the pathogenesis of muscular dystrophies.
Figure 1 Phenotypic characterization of the DGC mutants and eg28. a, Still pictures were taken immediately after animals had been transferred (stimulated condition) to a new plate seeded with Escherichia coli (OP50). snf-6 mutants rescued with a wild-type copy of the snf-6 gene show wild-type locomotion, whereas DGC mutants and wild-type animals treated with 1 mM aldicarb show the characteristic exaggerated bending (arrowhead) of the anterior body and head. b, Still pictures of wild-type and eg28 mutant animals from movies (Supplementary videos 5 and 6) showing locomotory changes before and after mechanical stimulation. Arrowhead, point of contact; t, time before or after stimulation.
Figure 2 snf-6 encodes a sodium-dependent neurotransmitter transporter. a, Genetic and physical maps of relevant region. +, phenocopy of eg28; -, no phenocopy of eg28. b, Rescue of snf-6 phenotypes with transgenes. Open box, origins of promoters; grey boxes, translated snf-6 coding sequence; parenthesis, number of rescued and tested transgenic lines; +, snf-6 phenotype; -, wild type. c, Predicted amino acid sequence of snf-6 and mutation sites. Asterisk, consensus glycosylation site; underline, transmembrane domain; boxed, PDZ recognition sequence. d, GFP reporter fused to 3 kilobases of DNA upstream of the snf-6 translation start site and the first two exons is expressed in muscles.
Figure 3 Biochemical characterization of SNF-6 by uptake assays. Results are means s.e.m. from triplicate trials with a stably transfected cell line expressing SNF-6 after subtracting the non-specific uptake from triplicate trials of a control cell line. a, b, Saturation curve of acetylcholine (a) and choline (b). Insets, Eadie–Hofstee transformation of saturation data. V, velocity; [S], substrate concentration. c, d, Sodium-dependent uptake of acetylcholine (c) and choline (d). The cells were incubated for 15 min with 10 nM acetylcholine or 20 nM choline in assay buffer containing either NaCl (control) or KCl. Values are expressed as a percentage of control.
Figure 4 SNF-6 is important for muscle integrity. a, Interaction of SNF-6 with STN-1. Full, full-length SNF-6; 3, SNF-6 lacking three C-terminal amino acids; IP, immunoprecipitation; IB, immunoblot; arrowhead, signals for Flag or HA epitopes; open arrowhead, Ig heavy chain. b, Localization of GFP::SNF-6 in mutants (48 h after L4). egIs2, egIs2[Pmyo-3::GFP::snf-6, pRF4(rol-6d)]; arrowheads, muscle arms; arrow, ends of muscle arms where NMJs occur. Fluorescent intensity comparison in snf-6;egls2 (NMJs, 42.24 3.30; membrane area, 26.04 1.01, P < 0.05). c, Four-day-old adults failing to move at least three body lengths after stimulation were scored as paralysed (snf-6, n = 64; hlh-1, n = 44; hlh-1;snf-6, n = 46; dyb-1;hlh-1, n = 56). d, Muscle organization in mutants. Arrowheads, degenerated muscle fibres. Scale bars, 10 µm.
Figure 5 snf-6 mutants exhibit enhanced evoked synaptic responses. a, c, Recordings from muscles of wild-type, snf-6, ace-1 and snf-6;ace-1 animals in response to five depolarizing stimuli delivered to the ventral nerve cord (20 Hz). b, d, The amplitude of each evoked response in the train was normalized to the initial response amplitude for each recording: comparison of normalized data for fifth evoked response in b (wild-type (squares), 23.5 1.9%, n = 17; snf-6 (circles), 35.5 2.8%, n = 8, P < 0.01) and d (ace-1 (squares), 16.4 2.7%, n = 5; snf-6;ace-1 (circles), 50.6 1.86%, n = 5, P < 0.01). All statistically derived values are given as means s.e.m.
